The present invention is directed to a device and a method of preparing vertebrae submucosa-derived collagenous matrices in combination with pre-selected cell population as tissue graft construct for the use in the repair of damaged or diseased tissues. The collagenous matrices for use in accordance with the present invention comprise highly conserved collagens, glycoproteins, proteoglycans, and glycosaminoglycans in their natural configuration and natural concentration. The extracellular collagenous matrix for use in this invention is derived from submucosal tissue of a warm-blooded vertebrae.
In accordance with the present invention the submucosa is isolated from warm-blooded vertebrae tissues including the alimentary, respiratory, intestinal, urinary or genital tracts of warm-blooded vertebraes. The preparation of intestinal submucosa is described and claimed in U.S. Pat. No. 4,902,508, the disclosure of which is expressly incorporated herein by reference. Urinary bladder submucosa and its preparation are described in U.S. Pat. No. 5,554,389, the disclosure of which is expressly incorporated herein by reference.
Stomach submucosa has also been obtained and characterized using similar tissue processing techniques. Such is described in U.S. Pat. No. 6,696,270 titled GASTRIC SUBMUCOSAL TISSUE AS A NOVEL DIAGNOSTIC TOOL issued Feb. 24, 2004. Briefly, stomach submucosa is prepared from a segment of stomach in a procedure similar to the preparation of intestinal submucosa. A segment of stomach tissue is first subjected to abrasion using a longitudinal wiping motion to remove the outer layers (particularly the smooth muscle layers and the luminal portions of the tunica mucosa layers. The resulting submucosa tissue has a thickness of about 100 to about 200 micrometers, and consists primarily (greater than 98%) of a cellular, eosinophilic staining (H&E stain) extracellular matrix material.
Preferred submucosal tissues for use in accordance with this invention include intestinal submucosa, stomach submucosa, urinary bladder submucosa, and uterine submucosa. Intestinal submucosal tissue is one preferred starting material, and more particularly intestinal submucosa delaminated from both the tunica muscularis and at least the tunica mucosa of warm-blooded vertebrae intestine.
As a tissue graft, submucosal tissue undergoes remodeling and induces the growth of endogenous tissues upon implantation into a host. It has been used successfully in vascular grafts, urinary bladder and hernia repair, replacement and repair of tendons and ligaments, and dermal grafts. The preparation and use of submucosa as a tissue graft composition is described in U.S. Pat. Nos. 4,902,508; 5,281,422; 5,275,826; 5,554,389; and other related U.S. patents. When used in such applications the graft constructs appear not only to serve as a matrix for the regrowth of the tissues replaced by the graft constructs, but also promote or induce such regrowth of endogenous tissue.
Common events to this remodeling process include: widespread and very rapid neovascularization, proliferation of granulation mesenchymal cells, biodegradation/resorption of implanted intestinal submucosal tissue material, and lack of immune rejection. The use of submucosal tissue in sheet form and fluidized forms for inducing the formation of endogenous tissues is described and claimed in U.S. Pat. Nos. 5,281,422 and 5,275,826, the disclosures of which are expressly incorporated herein by reference.
Submucosal tissue can be obtained from various sources, including intestinal tissue harvested from animals raised for meat production, including, for example, pigs, cattle and sheep or other warm-blooded vertebraes. This tissue can be used in either its natural configuration or in a comminuted or partially digested fluidized form. Vertebrae submucosal tissue is a plentiful by-product of commercial meat production operations and is thus a low cost cell growth substrate, especially when the submucosal tissue is used in its native layer sheet configuration.
The submucosa tissue graft constructs prepared in accordance with the present invention are a substantially acellular matrix that provides a superior cell growth substrate resembling the matrix environment found in vivo. The natural composition and configuration of submucosal tissue provides a unique cell growth substrate that promotes the attachment and proliferation of cells.
It has been reported that compositions comprising submucosal tissue of the intestine of warm-blooded vertebraes can be used as tissue graft materials in sheet or fluidized form. U.S. Pat. No. 4,902,508 describes tissue graft compositions that are characterized by excellent mechanical properties, including high compliance, a high burst pressure point, and an effective porosity index. These properties allow such compositions to be used for vascular and connective tissue graft constructs. When used in such applications the preferred graft constructs serve as a matrix for the in vivo regrowth of the tissues replaced by the graft constructs. U.S. Pat. No. 5,275,826 describes use of fluidized forms of vertebrae submucosal tissues as injectable or implantable tissue grafts.
The present invention is directed to preparation of submucosa tissue graft constructs as an implantable or injectable tissue graft construct. The improved tissue graft constructs are prepared by seeding the submucosal tissue in vitro with pre-selected or predetermined cell types prior to implanting or injecting the graft construct into the host.
The intestines of warm-blooded vertebraes are utilized for various purposes. For example, the small intestines of warm-blooded vertebraes are used in human food preparation for casings for sausages as well as in medical applications. The submucosa layer or inner layer of the small intestines is utilized for various purposes in medical applications as described herein, and therefore the processing of the inner layer or submucosa layer of the small intestines is of considerable interest. One of the initial steps in the preparation of small intestine submucosa is to slit and clean the submucosa layer of the small intestines. Cleaning of the intestines involves a scraping action. This scraping action may be called “despooging”. Business demands may require that the intestines be slit and despooged at the slaughterhouse in order to reduce cycle-time and labor costs.
The cleaning of the small intestines is typically done first by washing the inside of the small intestines with, for example, water. While the washing or cleaning of the small intestines with water may be sufficient for the use of the small intestines as a casing material for sausage, additional cleaning may be necessary when utilizing the small intestine submucosa layer for medical purposes. When utilized for medical purposes, subsequent processing in addition to the cleaning of the small intestine submucosa layer with water may be required. Such cleaning of the small intestines may require a scraping action on the intestinal layer. The intestines are split and then the inside layer of the intestines is scraped. Subsequent processing of the small intestine submucosa layer requires two separate operations. The first operation is to longitudinally slit the intestines and the second step involves a scraping action to scrape the small intestine inside layer while washing the layer. This process is known as “despooging”. Currently available equipment has required that the two operations be performed for slitting and despooging.
Referring now to FIGS. 2 and 3, prior art equipment, a slitter 1, for slitting and despooging the small intestine submucosa layer is shown. The stationary slitter 1, as shown in FIGS. 2 and 3, is large and not easily portable. The stationary slitter 1 measures approximately 36″×26″×18″. The stationary slitter 1 utilizes gears with a chain drive and electronic speed controls. The stationary unit requires the use of foot and hand controls which are difficult for the operator to synchronize during use. The unit has required the awkward holding of two acrylic plates and a precise angle for the cleaning steps. A concern with the stationary splitter was an exposed, unguarded scalpel blade.